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Abstract

Background

Endocrine FGF19 and FGF21 exert their effects on metabolic homeostasis through fibroblast
growth factor receptor (FGFR) and co-factor betaKlotho (KLB). Ileal FGF19 regulates
bile acid metabolism through specifically FGFR4-KLB in hepatocytes where FGFR1 is
not significant. Both FGF19 and FGF21 activate FGFR1-KLB whose function predominates
in adipocytes. Recent studies using administration of FGF19 and FGF21 and genetic
ablation of KLB or adipocyte FGFR1 indicate that FGFR1-KLB mediates the response of
adipocytes to both FGF21 and FGF19. Here we show that adipose FGFR1 regulates lipid
metabolism through direct effect on adipose tissue and indirect effects on liver under
starvation conditions that cause hepatic stress.

Methods

We employed adipocyte-specific ablations of FGFR1 and FGFR2 genes in mice, and analyzed
metabolic consequences in adipose tissue, liver and systemic parameters under normal,
fasting and starvation conditions.

Results

Under normal conditions, the ablation of adipose FGFR1 had little effect on adipocytes,
but caused shifts in expression of hepatic genes involved in lipid metabolism. Starvation
conditions precipitated a concurrent elevation of serum triglycerides and non-esterified
fatty acids, and increased hepatic steatosis and adipose lipolysis in the FGFR1-deficient
mice. Little effect on glucose or ketone bodies due to the FGFR1 deficiency was observed.

Conclusions

Our results suggest an adipocyte-hepatocyte communication network mediated by adipocyte
FGFR1 that concurrently dampens hepatic lipogenesis and adipocyte lipolysis. We propose
that this serves overall to mete out and extend lipid reserves for neural fuels (glucose
and ketone bodies), while at the same time governing extent of hepatosteatosis during
metabolic extremes and other conditions causing hepatic stress.

Keywords:

Background

FGF19 (FGF15 in mice) and FGF21 are circulating endocrine factors that affect metabolism
and metabolic diseases including obesity and diabetes [1-4]. FGF19 fluctuates with normal feeding-fasting cycles and is induced in the ileum
by postprandial bile acids and ligands of the farnesoid X receptor (FXR) [5-7]. In contrast, FGF21 is induced in the liver in response to liver perturbation [8-11] and during metabolic extremes that include those induced by starvation and obesity
[12-14]. Normally hepatic expression and blood level of FGF21 are low and vary widely among
individuals, but become consistently elevated during starvation [13].

Genetic deletion experiments have revealed that FGF19 regulates hepatic cholesterol/bile
acid synthesis specifically through hepatic FGFR4 in partnership with transmembrane
co-receptor KLB [6,15,16]. The direct tissue target and FGFR isotype that mediate FGF21 action and metabolic
effects of FGF19 in addition to hepatic bile acid metabolism are unclear. Recently
we showed that in addition to FGFR4-KLB, FGF19 also binds with high affinity and activates
FGFR1-KLB [17,18]. In contrast, FGF21 binds with high affinity and activates only FGFR1-KLB. FGFR4
is the predominant FGFR isotype expressed in hepatocytes whereas FGFR1 is expressed
at low to negligible levels [18-20]. FGFR1 is expressed along with KLB prominently in adipocytes where other FGFRs including
FGFR4 are very low or absent [18,19]. In view of the FGFR-KLB binding profile for FGF19 and FGF21, FGFR1 in adipocytes
is a candidate target for FGF21 specifically, while both FGFR1 in adipocytes and FGFR4
in hepatocytes are targets for FGF19. Based on response of signal transduction indicators
downstream of FGFR, adipose tissue specifically responds to infusions of FGF21 relative
to liver while both tissues are responsive to FGF19. The responses in adipocytes were
abrogated in mice deficient in adipose FGFR1 or KLB [18].

Manipulation of expression or pharmacological administration of FGF19 or FGF21 protein
in mice has major impact on metabolic diseases and pathways in hepatocytes in addition
to adipocytes [14,21-24]. A collaborative investigation in parallel to this study with our mouse model deficient
in adipose FGFR1 reveals that, under conditions of diet-induced obesity (DIO) and
FGF21 administration, adipose tissue expressing FGFR1-KLB accounts for nearly the
entirety of beneficial metabolic effects of FGF21 in vivoa. Under dietary restriction such as fasting and starvation conditions, FGF21 was proposed
as a starvation hormone and a paracrine factor acting on liver [13,14]. However, it is unclear whether the observed metabolic effects in liver under these
conditions relative to normal fed state in these two tissues are a direct consequence
of FGF-activated FGFR-KLB signaling in the respective tissue, or an indirect consequence
in the other through systemic inter-organ communication. In this report, we analyzed
under normal and starvation conditions the impact of adipocyte-specific deletion of
FGFR1 on metabolic parameters associated with adipose tissue and liver. The deletion
caused little change in adipocyte metabolic gene expression at the transcription level,
but instead caused elevation in expression of predominantly lipogenic genes in the
liver. The effects of the adipocyte FGFR1 deficit on hepatic lipid metabolism were
particularly evident under the metabolic extreme of starvation. From the observations,
we conclude that liver is a major indirect response organ to FGFR1 signaling in adipocytes.
Adipocyte FGFR1 serves to underpin a systemic communication between hepatocytes and
adipocytes mediated by FGF21 production from hepatocytes under stress that signals
through adipose FGFR1. Flux of free fatty acids and adipokines from adipocytes regulated
by FGFR1 in turn communicates back to liver to limit hepatic lipogenic gene expression
and extent of hepatic steatosis and associated stress. Under these conditions, FGFR1
concurrently dampened lipolytic activity in the adipocytes. We propose an axis of
hepatocyte-adipocyte cooperation mediated by hepatocyte FGF21 and adipocyte FGFR1
that serves to protect and mete out lipid reserves systemically while protecting the
liver against excessive steatosis and damage under metabolic extremes and general
hepatic stress.

Materials and methods

Animals

The FGFR1-floxed (FGFR1lox/lox, referred to hereafter as FGFR1Fx or control), FGFR2-floxed (FGFR2Fx) and aP2-Cre mice have been described [25-27]. These mice were backcrossed to C57BL/J6 background for more than 5 generations.
Mice with deficiency in FGFR1 and FGFR2 specifically in adipocytes were generated
by crossing the FGFR1Fx and FGFR2Fx mice with aP2-Cre mice as described (FGFR1lox/loxaP2Cre and FGFR2lox/loxaP2Cre, referred to hereafter as FGFR1Cn and FGFR2Cn), respectively [18]. Experimental animals were male. The 12 h fast began at 6:00 PM with 70 percent of
the time duration in dark cycle when murine daily food intake occurs. The 48 h fast
began at 6:00 AM when the light cycle started. All mice were housed in the Program
of Animal Resources in the Institute of Biosciences and Technology, and were handled
in accordance with the principles and procedure of the Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by the Institute of Biosciences and Technology
Institutional Animal Care and Use Committee (IBT IACUC) with protocol #10022 entitled
“BetaKlotho-FGFR in the liver” and #09008 entitled “IBT-Mouse Models”.

Analysis of gene expression

Total RNA isolation and quantitative PCR were done as described [18]. Representative liver tissues were from the left lobe, and gonadal adipose tissues
were used as representative for the adipose tissue, from the number of mice as indicated.

Cellular fractionation of adipose tissue

Adipocytes were separated from the stromal-vascular (SV) fraction by a modified Rodbell
method [28]. Adenosine was added to suppress lipolysis. About 2 gram of fat from male mice at
ages of eight to ten weeks were minced into 2–3 mm diameter pieces and dissociated
with 3 mg/ml collagenase in Krebs–Ringer-Hepes (KRH) buffer [129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1 mM CaCl2, 1.2 mM MgCl2, 2.8 mM glucose and 10 mM Hepes (pH7.4)], at 37°C with slow shaking for 1 h. After
filtration through 250 μm gauze meshes, the adipocyte and SV fractions were separated
by centrifugation at 200 g for 5 min. Adipocytes were carefully harvested from the
top fat cake layer and the SV fraction was from the pellet on the bottom.

Liver and adipose tissue histology

Portions of freshly isolated liver and adipose tissues were fixed with Histochoice
Tissue Fixative MB (Amresco, Solon, OH) and paraffin-embedded. Tissue sections were
stained for general pathological examination with hematoxylin and eosin. In addition,
liver tissue was processed in Neg-50 frozen section medium. Lipid droplets were revealed
by staining the frozen section with Oil Red O at 60°C for 8 min. After washing with
85% isopropanol, the sections were further counterstained with hematoxylin.

Adipokine array

Equal amounts of serum samples from FGFR1Cn or FGFR1Fx mice under different diet restriction
conditions as indicated, were used to perform antibody array analysis against selected
adipokines following the manufacturer’s protocol (R&D Systems, Minneapolis, MN).

Serum activities of liver enzymes ALT and AST

The blood samples from FGFR1Fx and FGFR1Cn mice (n=6 each group) were collected after
fed or fasted for 48 h. The activities of liver blood enzymes alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) were measured with an enzymatic activity
assay kit and human based serum calibrator (DC-CAL) (Sekisui diagnostics, Charlottetown,
PE Canada).

Statistical analysis

Experiments were reproduced three times independently with triplicates for each experiment.
A representative of three or more experiments may be shown in micrographs. Where indicated,
the mean and standard deviation (SD) was determined. Comparisons between different
genotype groups were performed with the unpaired t test. Values were deemed to be statistically significantly different at p≤0.05.

Results

Ablation of FGFR1 in adipocytes causes changes in hepatic, but not adipocyte metabolic
gene expression

Mice deficient in FGFR1 in adipocytes (FGFR1lox/loxaP2Cre or FGFR1Cn) were generated from mice with the floxed FGFR1 gene (FGFR1lox/lox or FGFR1Fx) [26] and mice expressing Cre recombinase driven by the promoter of adipocyte fatty acid
binding protein aP2 [25,29]. LacZ staining in mice with both aP2-Cre+: ROSA26R+ alleles [27] as compared to the control indicated that the aP2 promoter was active in a majority
of adipocytes (Additional file 1: Figure S1A and B). When compared to liver, muscle and kidney, the expression of
FGFR1 in total adipose tissue of the FGFR1Cn mice was reduced by 50 percent (Additional
file 1: Figure S1C). To determine efficacy of the FGFR1 ablation specifically in the adipocyte
fraction, adipose tissue was fractionated into mature adipocyte and the stromal-vascular
(SV) fractions based on aP2 expression. FGFR1 mRNA in the adipocyte fraction of the
FGFR1Cn mice was reduced to less than 5 percent that of wildtype or FGFR1lox/lox control mice and to 50 percent in the stromal-vascular fraction (Additional file
1: Figure S1D). Expression of FGFR2 in mature adipocytes was unchanged in the FGFR1Cn
mice. Expression of co-factor KLB in mature adipocytes was over 15 times that in the
stromal-vascular fraction and was unaffected by the absence of FGFR1.

Additional file 1.Figure S1. Specificity and efficiency of FGFR1 ablation in adipose tissue using the aP2 promoter.
(A) Expression of the aP2 promoter in adipose tissue. The male reproductive complexes
with attached gonadal adipose tissues in LacZ ROSA26R reporter mice (aP2Cre-) and
the reporter mice crossed with aP2Cre mice (aP2Cre+) were analyzed by LacZ staining
(blue). (B) Expression of the aP2 promoter in adipocytes. A paraffin-embedded section
of the LacZ-stained (blue) gonadal fat tissue from (A) showed that more than 90 percent
of adipocytes were positive for the aP2Cre recombinase activity. Tissue was counterstained
with H&E. (C) FGFR1 mRNA expression among different tissues in FGFR1Fx and FGFR1Cn
mice. FGFR1 expression was assessed by quantitative PCR. Total white adipose tissue
(WAT) exhibited a 50% reduction of FGFR1 expression in the FGFR1Cn mice. *p<0.05
(n=5). (D) Relative expression of FGFR1, FGFR2 and KLB in mature adipocyte and the
stromal-vascular (SV) fractions of adipose tissue. a: significant difference between
adipocytes and sv fractions. b: significant difference between FGFR1Fx and FGFR1 Cn
in the same adipocytes fraction or sv fraction. Data are the mean ± SD (n = 7-8),
* p<0.05. Figure S2. Relative serum levels of FGF21 in the FGFR1Fx and FGFR1Cn mice
at fed or fasted stages. The relative serum levels of FGF21 were measured by adipokine
array kit (R&D systems) according to product manual. Sera were pooled from 3 mice
(50 ul each mouse) for each genotype at fed state or after starved for 48 h. The dot
blot membranes were analyzed for FGF21 antigen levels (A) and the relative intensity
of spot was determined by densitometry (B). Figure S3. Lack of effect of the adipocyte
FGFR1 deficiency on serum metabolic parameters in the fed state. Sera were collected
from mice fed ad libitum within one hour after the start of the light part in the
light–dark cycle. Imposed fasting for 4 h yielded similar results. The indicated parameters
were assessed as described in text Figure 5. Data are the mean ± SD (n= 10), p<0.05 for all tests. Figure S4. Lack of effect
of the adipocyte FGFR1 deficiency on metabolic gene expression in adipose tissue.
Expression of the indicated genes as described in Additional file 1: Table S1 in FGFR1Fx and FGFR1Cn mice was assessed by quantitative PCR after 4 h
fasting or 48 h starvation. The expression levels were standardized relative to those
of FGFR1Fx mice with 4 h fasting, which were assigned a value of 1. Data are the mean
± SD (n= 10), p<0.05 for all tests. Figure S5. Effects of adipose FGFR1 deficiency
on the expression of oxidative stress markers Ucp2 and Nrf2. mRNA levels for hepatic
Ucp2 and Nrf2 were determined by quantitative PCR analyses. The expression level is
relative to the FGFR1Fx under normal fed condition, which is considered as an arbitrary
unit 1. Data are the mean ± SD of 6 mice for each group, p<0.05. Figure S6. Effects
of adipose FGFR1 deficiency on serum enzyme activities for liver ALT and AST. Blood
is collected from FGFR1Fx and FGFR1Cn mice after food starvation for 48 h, and serum
is used to measure enzyme activities of liver-derived ALT and AST as a result of liver
injury and diseases. Data are the mean ± SD of 6 mice for each group, * p<0.05. Table
S1. Metabolic genes analyzed in expression analyses.

The absence of FGFR1 in the adipocytes precipitated significant changes at the mRNA
level in expression of hepatic genes involved in lipid metabolism. The shifts predominantly
were relative increases in expression of those associated with lipogenesis (Figure
1A). The mRNA for membrane fatty acid transporter CD36 was increased by a dramatic 6
fold accompanied by an increase of 3 fold in the key lipogenic transcriptional regulator
PPARγ. Acyl-CoA oxidase (AOX) expression, an enzyme involved in polyunsaturated fatty
acid genesis and degradation of very long chain FFA, was elevated by 2.5 fold. However,
expression of two genes coding for factors associated with fatty acid oxidation, MTP
and MCAD, was also increased (Figure 1B). No changes were observed in genes associated with cholesterol/bile acid metabolism
or gluconeogenesis (Figure 1C). Notably, a 3 fold increase in hepatic and systemic FGF21 expression was observed
(Figure 1D, Additional file 1: Figure S2). This was coincident with significant increases in expression of reported
regulators for hepatic FGF21 expression, PPARα and PGC1α [14,23] in response to absence of adipocyte FGFR1 (Figure 1D).

Figure 1.Effects of adipocyte FGFR1 deficiency on hepatic metabolic gene expression during
normal feeding-fasting. Relative expression of the indicated genes in the liver (Additional file 1: Table S1) involved in lipogenesis (A), fatty acid transport and oxidation (B), gluconeogenesis and bile acid/cholesterol metabolism (C) and FGF21 transcriptional regulation (D), were assessed by quantitative PCR in control and adipocyte-deficient FGFR1 mice
in normal fed state or after a 4 h fast. Expression in control FGFR1Fx mice was assigned
a value of 1. Data are the mean ± SD of 10 mice with p<0.05 for all tests.

These adipocyte FGFR1-dependent shifts in hepatic metabolic gene expression occurred
in the absence of overt changes in body weight, adiposity and morphology of adipocytes
or hepatocytes (Figure 2A-2D), in serum metabolic parameters including triglycerides, NEFA, glucose and ketone
bodies (Additional file 1: Figure S3) and insulin (not showed), and in expression at the mRNA level of a variety
of genes in adipose tissue that code for metabolic parameters (Additional file 1: Figure S4). This indicates that in normal physiology, adipocyte FGFR1 underlies
systemic signals from adipocytes to hepatocytes that impact hepatic metabolic gene
expression that is in largest part depression of those involved in lipogenesis.

The absence of adipocyte FGFR1 results in increase of starvation-induced hepatic steatosis
coincident with increases in hepatic lipogenic gene expression

No changes in hepatic lipid content or overt hepatocyte morphology were observed due
to the absence of adipocyte FGFR1 under normal feeding conditions. Starvation is an
extreme metabolic condition that imposes stress on the liver and is evident by development
of hepatic steatosis. Under starvation condition, candidate activating endocrine ligands,
FGF21 [13,14] and FGF19 [5,7], are also induced to sustained elevated levels. Therefore, we imposed a prolonged
48 h starvation on mice to test for additional phenotypes in liver, adipose tissue
and serum. The deficit of adipocyte FGFR1 increased the severity of the hepatic steatosis
(Figure 3A). A 1.5 fold increase in hepatic triglyceride (TG) content coincident (Figure 3B) with elevation of expression of hepatic genes specifically involved in lipid metabolism,
not glucose or ketone body metabolism, was also evident in the deficient mice. The
changes in hepatic gene expression due to absence of adipocyte FGFR1 are still tightly
clustered around lipogenic genes similar to observations during normal feeding conditions
(Figure 4). Notable differences caused by the adipocyte FGFR1 deficiency between normal feeding
and the 48 h starvation conditions were elevations of SREBP1c and SCD1 by 2.5 and
6 fold (Figure 4A), respectively, and lack of increases in fatty acid degradative factors MTP and MCAD
(Figure 4B) and in regulators for bile acids metabolism and gluconeogenesis (Figure 4C). The increase of SREBP1c, a fatty liver and hepatic ER stress marker [30], indicates that an increase of hepatic stress may accompany the exaggerated lipogenesis
and steatosis caused by the metabolic stress imposed by starvation and adipose FGFR1
deficit. Prolonged starvation significantly increases hepatic expression of Nrf2 and
Ucp2 but not MnSOD, which are oxidative stress markers; however, an effect of the
adipose FGFR1 deficit was not statistically significant (Additional file 1: Figure S5). A lack of increases in MTP and MCAD in the liver under prolonged starvation
is also consistent with the increase of hepatic lipogenesis and steatosis in the adipose
FGFR1 deficient mice. The elevation of PPARα and hepatic FGF21 mRNAs caused by the
loss of adipocyte FGFR1 observed during normal feeding remained intact in the starvation
conditions, while the effect on PGC1α expression and systemic FGF21 was masked or
at a maximal level (Figure 4D, Additional file 1: Figure S2). These results suggest that when liver is under metabolic stress induced
by starvation and steatotic conditions, adipocyte FGFR1 and its activating ligands
underlie signals from adipocytes to hepatocytes that overall dampen hepatic lipogenic
gene expression and limit extent of starvation-induced hepatic steatosis.

Figure 3.Increased starvation-induced fatty liver and lipid content in mice deficient in adipose
FGFR1. (A) Appearance of liver tissue. Conventional hematoxylin and eosin (H&E) staining on
liver sections revealed the elevated characteristic vacuoles induced by lipid accumulation.
Staining by Oil Red O revealed an increase in the number and size of lipid droplets.
(B) Quantitation of liver TG content. Data are the mean + SD (n=7-8). **p<0.005, and
p<0.05 for all other tests.

Figure 4.Increase in gene expression of hepatic lipogenic enzymes caused by absence of adipose
FGFR1 after starvation. Expression of the indicated hepatic genes as described in Figure 1 (Additional file 1: Table S1) was assessed by quantitative PCR in control and adipocyte-deficient FGFR1
mice after food restriction for 48 h. Expression level in FGFR1Fx mice was assigned
a value of 1. Data are the mean ± SD (n= 10), p<0.05 for all tests.

Serum NEFA and triglycerides, but not glucose or ketone bodies, are concurrently elevated
in starved mice deficient in adipocyte FGFR1

As noted earlier despite shifts in primarily hepatic lipogenic gene expression including
systemic FGF21, no changes in serum NEFA, triglycerides, glucose or ketone bodies
during normal feeding and even after a 24 h fast could be clearly detected as a consequence
of the adipocyte FGFR1 deficiency. However, imposition of the stress of starvation
conditions when hepatic steatosis is apparent caused a detectable rise in serum TG
and NEFA levels at 1.5 and 1.4 fold that of wildtype or FGFR1Fx mice (Figure 5A and B). In contrast, neither the depressed glucose levels (Figure 5C) nor elevated ketone bodies elicited by starvation (Figure 5D) changed between control and deficient mice. These starvation-dependent observations
on serum parameters elicited by the adipocyte FGFR1 deficiency are consistent with
the general elevation of specifically hepatic lipogenic genes and hepatic steatosis,
without effect on hepatic genes involved in glucose and ketone body metabolism. The
forced increase in NEFA level from adipocytes due to FGFR1 deficit under prolonged
starvation, likely in part resulted in the observed compensatory increases of hepatic
and serum FGF21 through hepatic PPARα, and the elevated hepatic lipogenesis and steatosis
as well.

Figure 5.Elevated serum TG, NEFA and adipose lipase activity induced by starvation stress in
mice deficient in adipocyte FGFR1. (A) Serum triglycerides, (B) NEFA, (C) glucose and (D) ketone bodies were analyzed in FGFR1Cn and FGFR1Fx mice after food restriction for
12 and 48 h. Data are the mean ± SD (n = 10), ** p<0.005, and p<0.05 for all other
tests. (E) Adipocyte total lipase activity. Rate of lipase activity was assessed by fluorescence
as described in Materials and Methods in mice starved for 48 h. Data are the mean
± SD (n = 6).

To further quantitatively determine the hepatic stress level resulting from exaggerated
hepatic steatosis due to the adipose FGFR1 deficiency, we analyzed blood activities
of the two liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase
(AST). Serum ALT and AST are indicators for pathological changes in hepatocytes and
liver damage/injury and diseases. There is no significant change in serum activities
of ALT and AST upon adipose FGFR1 deficiency in the normal fed state. Starvation increases
the activities of serum ALT at about 8% and 22% and AST at about 49% and 80% in the
FGFR1Fx and FGFR1Cn mice, respectively. These results demonstrate that adipose FGFR1
deficiency significantly elevates the serum enzyme activities of both ALT and AST,
indicating significant exaggeration of liver stress levels upon adipose FGFR1 deficiency
in starvation (Additional file 1: Figure S6).

To determine whether adipocytes participate directly in the starvation-dependent phenotypic
effects of the adipocyte FGFR1 deficiency, we again tested for changes in adipocyte
gene expression. Regulation of expression of adiponectin by adipose FGFR1 signaling
has been demonstrated a. However, little effect of the adipocyte FGFR1 deficit on expression of many other
metabolic genes coding for major metabolic enzymes and regulators at the mRNA level,
including hsl and atgl, in adipose tissue was observed, similar to normal feeding
and short-term fasting (Additional file 1: Figure S4). Notably, the rate of lipoprotein lipase enzyme activity in adipocyte
extracts from the FGFR1Cn mice was about 1.45 times that of control mice (Figure 5E). The change in adipose lipase enzyme activity but no effect at the mRNA level suggested
a post-transcriptional regulation of lipase activity by adipocyte FGFR1 signaling.
This result indicates that under starvation conditions, when an activating FGF ligand
is sufficient, adipocyte FGFR1 serves to dampen lipolysis in the adipocytes while
concurrently eliciting signals that dampen lipogenesis and steatosis in hepatocytes.

Ablation of adipose FGFR2 does not elicit the hepatic response elicited by the FGFR1
deficiency

FGFR2 is expressed at the mRNA level in adipocytes and adipose tissue (Additional
file 1: Figure S1D). To address whether FGFR2 played a similar role to FGFR1 in adipose
tissue, we prepared a mouse line deficient in adipocyte (FGFR2 FGFR2lox/loxaP2Cre or FGFR2Cn) following the same approach as described for FGFR1. In agreement with
a previous report [31], the FGFR2 deficient mice exhibited no overt abnormalities in development and cellular
homeostasis. Moreover, the elevation in serum TG content and starvation-induced hepatic
steatosis caused by the adipocyte FGFR1 deficiency was not observed in animals deficient
in adipocyte FGFR2 (Figure 6A and B). This indicates that adipocyte FGFR1, but not FGFR2 underpins the phenotypes
in lipid metabolic homeostasis observed in this study.

Figure 6.FGFR2 deficiency in adipocytes exerted no comparable effect on starvation-induced
fatty liver. (A) Histology of liver tissue from adipocyte-deficient FGFR2 (FGFR2Cn) mice was compared
to that from the FGFR1Cn and FGFR2Fx mice in both fed and 48 h starvation states at
two magnifications. The enhanced steatotic vacuolization observed in FGFR1Cn mice
was not apparent in the FGFR2Cn mouse livers. (B) Quantitation of liver TG content from FGFR2Fx and FGFR2Cn mice. Data are the mean
± SD (n= 10), p<0.05 for all other tests.

Discussion

Here we deduce from results of gene ablation, that under normal dietary condition,
FGFR1 in adipocytes normally underlies signals from adipocytes that restrict hepatic
expression levels of a subset of genes involved in lipid metabolism. The subset is
in largest part genes associated with hepatic lipogenesis. This occurs without apparent
changes in adipocyte metabolic gene expression, expression of hepatic genes involved
in glucose metabolism, blood levels of glucose and lipids and morphology of adipocytes
and hepatocytes.

In the presence of transmembrane co-receptor KLB, both FGF19 and FGF21 bind with high
affinity and activate FGFR1 [17,18,20]. In contrast to FGF19, FGF21 fails to bind and activate FGFR4-KLB that is the predominant
FGFR in hepatocytes. FGF21 is specific for the FGFR1-KLB complex which is the predominant
FGFR isotype in adipose tissue [18,20,32]. This differential specificity limits the direct action of FGF21 to adipose tissue
relative to liver, in contrast to the activity of FGF19 which acts potentially on
both adipocytes via FGFR1 and hepatocytes via FGFR4. The activation of adipose tissue
by FGF21 through FGFR1 was suggested by genetic deletion of FGFR1 in adipocytes in
mice used in the current study [18], and this tissue- and molecule-specific actions of FGF21 underlies the breadth of
its in vivo beneficial effects on treatment of obesity and diabetes a. FGF19 is a diurnal hormone that fluctuates during normal feeding [7]. FGF21 comes into play after prolonged periods of caloric restriction or hepatic
perturbation by conditions such as steatosis or chemical damage [8-14]. This suggests that during normal feeding, ileal FGF19 is likely the activating endocrine
FGF for adipocyte FGFR1. Thus abrogation of FGF19-adipocyte FGFR1 signaling may underlie
effects of the FGFR1 deficiency on hepatic lipid gene expression under normal conditions.
Such indirect effects of FGF19 on hepatic lipogenic gene expression through adipocyte
FGFR1-KLB are in addition to the direct effects on hepatic bile acid [6,15,16] and lipid metabolism mediated by FGFR4-KLB [9]. The impact of the changes in hepatic gene expression contributed by adipocyte FGF19-FGFR1
signaling during normal feeding on the hepatic contribution to overall metabolic homeostasis
remains to be determined. Notably, the deficiency of FGFR1 in adipocytes also caused
an increase in hepatic FGF21 without apparent effect on ileal FGF15 (Yang C, unpublished
data). This indicates that during normal feeding FGF19 working through adipocyte FGFR1
may suppress expression of hepatic FGF21. This may be the reason why normally FGF21
levels are low and variable, and only rise to high sustained levels during severe
metabolic extreme as starvation and other causes of hepatic stress or perturbation.

We used starvation conditions to impose metabolic stress on the liver that is evident
by overt hepatic steatosis. Under these conditions, we observed systemic and hepatic
metabolic consequences due to the adipocyte FGFR1 deficiency. We therefore deduce
that under conditions as starvation that causes severe hepatic stress, the role of
adipocyte FGFR1 and its cognate activating ligands underlies signals that restrict
adipocyte lipolysis and through adipocyte to hepatocyte signals concurrently restrict
extent of hepatic lipogenesis. These signals likely include free fatty acids and/or
adipokines such as adiponectin, as a result of changes in lipolysis and endocrine
function of adipose tissue governed by adipose FGFR1 signaling. Increased free fatty
acids from adipose tissue deficient in FGFR1 further enhance the expression of hepatic
FGF21 through its nuclear receptor PPARα, whose expression is also increased under
these conditions that may be due to change in adiponectin level [33,34]a. Increased free fatty acids also contribute to exacerbated hepatic steatosis. These
results suggest an inter-organ communication between adipose tissue and the liver
mediated by hepatic FGF21 and adipose FGFR1 through adipocyte signals that include
free fatty acids and/or adipokines. In view of the absence of overt phenotypic effects
during normal feeding beyond the changes in hepatic lipid gene expression, our results
suggest that adipocyte FGFR1 plays its most important physiological role under metabolic
extremes and other conditions that stress the liver. Such conditions induce sustained
and maximal levels of serum FGF21 [13,14] and FGF19 [5], and thus are conditions where endocrine activation of adipocyte FGFR1 would be maximal.
We propose that the phenotypes observed using starvation as a specific metabolic extreme
may reflect a general hepatic-adipocyte communication network, which is comprised
of hepatic FGF21 induced by general metabolic or other type of stress on the liver,
adipocyte FGFR1 and FGFR1-mediated metabolite and adipokine signals such as free fatty
acids and adiponectin or other yet to be identified factors back to the liver (Figure
7). In addition to starvation, an increase in hepatic FGF21 generally accompanies hepatic
stress-inducing conditions of obesity and chemical insult, infection and inflammation
[8-14,35,36]. This communication primarily governed by adipose FGFR1 serves to limit adipose lipolysis
and hepatic steatosis, liver stress and resultant damage under stressful or adverse
conditions.

A hallmark of the starvation-induced signaling axis from hepatic FGF21 to adipocyte
FGFR1 indicated by our study, is the concurrent changes in adipocyte lipolysis and
hepatic lipogenesis partitioned between the two organs. Under the starvation conditions,
the changes in lipid metabolism also appear relatively independent of glucose and
ketone body metabolism. Normally lipid metabolism including both lipolysis and lipogenesis
is tightly coupled to glucose and ketone body metabolism in overall metabolic homeostasis.
Moreover, overall lipolysis and lipogenesis are tightly coupled and inversely related
to each other. During the course of starvation, glucose levels progressively sink.
The uncoupling promoted by the hepatocyte-adipocyte axis may serve to slow down the
flow of lipids into glucose and ketone bodies. This extends critical lipid stores
for as long as possible for maintenance of brain fuels above critically low levels
until feeding resumes. This metabolic uncoupling promoted by the hepatic FGF21-adipocyte
FGFR1 axis may be of temporary benefit to the organism under conditions of not only
starvation, but also a variety of other conditions that are stressful to the liver
when overloaded beyond its routine role in metabolic homeostasis. Reversible hepatic
steatosis generally accompanies such conditions. The endocrine FGF-mediated hepatocyte-adipocyte
axis may also serve to limit hepatic steatosis before it becomes irreversibly damaging.

Conclusions

Adipocyte-specific deletion of FGFR isotypes, FGFR1 and FGFR2 show that specifically
adipose FGFR1 mediates indirect effects in liver that are most significant under starvation
conditions which causes hepatic stress and steatosis. This occurs through direct adipose
FGFR1-dependent restrictions on adipocyte lipolysis [37] and indirectly hepatic lipogenesis through systemic adipocyte to hepatocyte signals.
This communication also serves to attenuate extent of compensatory hepatic steatosis
that often occurs during hepatic stress. This adipohepatic communication cycle also
serves overall to mete out and extend lipid reserves for neural fuels (glucose and
ketone bodies) during metabolic extremes and other conditions causing hepatic stress.
This is particularly of benefit, possibly life-saving, during prolonged starvation
to preserve consciousness until feeding opportunity occurs. Since adipocyte FGFR1
is a selective target of FGF21 and an additional target of FGF19 in addition to hepatocyte
FGFR4, our results predict that this adipocyte-directed mechanism [38,39] may underpin the beneficial effects of endocrine ligands, FGF21 and FGF19, observed
under conditions of not only caloric restriction, but also excess as in obesity that
both cause metabolic perturbation in the liver.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CY, WLM and YL designed research. CY, MY, CJ and YL performed research. WH and FW
contributed reagents. CY, YL and WLM analyzed data. CY, WLM and YL wrote the paper.
All authors read and approved the final manuscript.

Acknowledgements

This work was supported by US Public Health Service grants DK56338 (Texas Medical
Center Digestive Diseases Center), Enhancement Grant from Texas A&M Health Science
Center, P50 CA140388 to WLM, and the Susan Komen Breast Cancer and the John S. Dunn
Research Foundations.